DIRECT LYSIS BUFFER AND THE DETECTION OF HIV-1 PLASMA VIREMIA

Abstract: Immunocapture of plasma HIV-1, coupled with direct lysis of the virions and a simplified method of reverse transcription and amplification of the HIV-1 cDNA by the Polymerase Chain Reaction (PCR) represents a rapid and highly sensitive method to monitor HIV-1 disease progression. This method is also less time and labor intensive than quantitative culture. In addition, the development of a method to directly lyse the immunocaptured virions and a simplified single step reverse transcription (RT)/PCR procedure eliminated the need for organic solvent extraction and reduced the number of steps in the procedure. A direct lysis buffer was formulated to isolate plasma HIV-1 RNA for direct use in the RT and PCR reactions, thus eliminating the need for organic solvent extraction and ethanol precipitation. This resulted in a significant saving of time needed to complete the assay and significantly reduces the possibility of contamination associated with PCR reactions. The immunocapture-RT/PCR assay was used to show that vertical transmission of HIV-1 from a mother to her child depended largely on factors other that viral load. Conversely, the plasma viral load played a significant role in transfusion associated transmission of HIV-1 infection. Finally, the detection and quantitation of plasma associated viral load by immunocapture-RT/PCR may provide an additional marker of disease progression and may aid in determining the efficacy of various HIV therapeutics.

Description: DIRECT LYSIS BUFFER AND THE DETECTION OF HIV-1 PLASMA VIREMIA FIELD OF THE INVENTION The present invention involves a process to disrupt virions and isolate the nucleic acid of the virus. In particular, the invention presents a direct lysis buffer involving a combination of detergent and Proteinase K to isolate nucleic acids. BACKGROUND OF THE INVENTION When the first cases of acquired immune deficiency (AIDS) were described in 1981 the causative agent of the disease was unknown. In 1983, a French laboratory headed by Luc Montagnier isolated the causative virus now known as human immunodeficiency virus type 1 (HIV-1). At or around the same time Robert Gallo and members of his laboratory, at the National Institutes of Health, reported having isolated the causative agent of AIDS. Within two years, diagnostic assays were developed and used to identify persons infected with HIV-1. These assays, which were developed to be both highly sensitive and specific, detected the presence of antibodies to HIV-1 in serum or plasma. Epidemiological studies found that the HIV-1 virus was mostly detected in specific populations, such as homosexual men and hemophiliacs, and that the main mode of transmission was through sexual contact or receipt of infected blood products. It was later found that IV drug users were a group at high risk of transmitting HIV because of the practice of sharing used needles (i.e., cross contamination of blood). The correlation between infection by HIV and exposure to infected blood, or the use of infected blood products, prompted the start of mandatory screening in blood banks to reduce and control the spread of viral infection. A system implementing the testing of all 20 million annual blood donor units and blood products was established. This routine testing significantly reduced the number of HIV-1 related cases due to either blood transfusions or receipt of processed blood products. At present, the risk of transfusion- associated HIV infection is estimated to be approximately 1 :250,000. Although the number of transfusion-associated transmissions of HIV have decreased, the number of AIDS related cases continue to grow throughout the world. As of 1992, the estimated number of people infected with the HIV-1 virus in the U.S and worldwide is at one million and ten million, respectively. In the U.S. alone, it is estimated that 40,000 new HIV-1 infections occur each year (Centers for Disease Control. HIV Prevalence Estimates And AIDS Case Projections For The United States: Report Based Upon A Workshop. MMWR 39:(no.RR-16) November 30, 1990). Of these 11.5% and 1.7% occur in women and adolescent children, respectively. The majority of female HIV cases are a result of either IV drug use or sexual contact with an HIV-infected partner. The majority of adolescent HIV-1 cases are a result of the vertical transmission of HIV-1 from the mother to her child. Worldwide, the rate of vertical transmission of HIV-1 is reported to be between 10% and 40% (The European Collaborative Study. Children Born To Women With HIV-1 Infection: Natural History And Risk Of Transmission. Lancet 1991;337:253-260; and Ryder, R.W., Nsa, W.N., Hassig, S.E., Behets, F., Rayfield, M. and Project SIDA. Perinatal Transmission Of The Human Immunodeficiency Virus Type 1 Infection To Infants Of Seropositive Women In Zaire. N. Engl. J. Ivied. 1989;320:1637-1642). Of the estimated 40,000 new cases of HIV-1 per year in the U.S., 1500 to 2000 infections will occur in newborns as a result of perinatal HIV-1 transmission. The fact that the number of AIDS-related cases increases each year is due in part to the characteristics of the infection. Determining ways of controlling its progression is vital if a means of reducing its spread is to be achieved. HIV-1 Genetic Structure and Replication HIV is a member of the retrovirus family. Retroviruses are characterized as having RNA as their genetic material and contain the unique enzyme reverse transcriptase (RT), which catalyzes the reverse transcription of the RNA genome into a DNA copy (cDNA). There are three subfamilies of retrovirus. HIV belongs to the lentivirus subfamily based on its structural and genetic properties. Typically, retroviral genomes are composed of between 9,000 and 10,000 base pairs and contain three structural genes that are characteristic to all retroviruses (gag, pol, and env). They contain unique sequences located at the 3' terminus of pol and env that code for regulatory proteins. Located at both the 5' and 3' ends of the genome are two identical sequences called long terminal repeats (LTR). The 5' LTR is critical for the expression of proviral DNA by the host's cellular transcription machinery . The HIV-1 RNA genome is composed of a total of 9,749 nucleotides, representing 9 genes (Haseltine, W.A., Wong-Stall, F. The Molecular Biology Of The AIDS Virus. Scientific American 1988;259:52-62). The genome contains the three characteristic structural genes and an additional six regulatory genes (tat, rev, vif, vpr, nef, and vpu). The gag and pol proteins are translated from full length transcript, while the env protein is translated from a spliced transcript. The gag gene is transcribed to give a full length RNA and translated to give a precursor polyprotein that is subsequently cleaved into three capsid proteins, which make up the major structural proteins of the virus core. The pol protein is actually part of a gag-pol precursor. The pol portion of the gene encodes the enzymes associated with the RNA inside the core of the virus, the protease, reverse transcriptase and integrase. The reverse transcriptase actually has three enzymatic functions, RNA dependent DNA polymerase, DNA dependent DNA polymerase and ribonuclease activity. The envelope gene (env) encodes a precursor protein, gp160, that is cleaved by a protease to make the extracellular glycoprotein gp120 and the transmembrane protein gp41. The gp120 protein is responsible for binding the virus to the cell surface CD4 receptor. The gp41 protein mediates syncytia formation and also assists in the penetration of the virus core into the interior of the cell (Sodrowski, J., Goh, W.C., Resen, S., Campbell, K., and Haseltine, W.A. Role Of The HTLV-III/LAV Envelope In Syncytium Formation And Cytopathicity. Nature 1986;322:470-474; and McCune, J.M., Rabin, L.B., Feinburg, M.B., Lieberman, M., Kosek, J.C., Reyes, G.R., and Weissman, I.L. Endoproteolytic Cleavage Of gp160 Is Required For The Activation of Human Immunodeficiency Virus. Cell 1988;53:55-67). The six additional genes regulate the production of viral proteins necessary for replication and assembly of the virus (Haseltine, above). The structure of HIV-1 resembles that of all retroviruses. It contains a cylindrical core which is made up of two gag proteins. Inside the core are two identical single stranded RNA molecules. Associated with the RNA genome are the enzymes reverse transcriptase, protease and integrase. The core is surrounded by an envelope derived from the host cell's plasma membrane. The surface of the membrane is studded with copies of the HIV-1 specific protein, gp120, which are noncovalently associated with the gp41 transmembrane protein. The infectious cycle of HIV begins when viral envelope proteins bind to the CD4+ molecule that is found on the host cell surface. The CD4+ molecule is typically found on T lymphocytes and macrophage/monocytes. The membranes of the virus and host cell fuse, and the core of the virus is injected into the host cell. Once the core is inside the host cell, the viral RNA genome is reverse transcribed into a cDNA copy. The RNA genome is then destroyed by the RT-associated enzyme Ribonuclease H, and the polymerase makes a second DNA copy using the cDNA copy as a template. This double stranded viral DNA migrates into the nucleus where it is integrated into the host cell's DNA by way of the viral protein integrase. Once integrated, the viral DNA is termed a provirus. The production of new virus particles, their release from the cell, and infection of new cells complete the cycle of HIV infection. Production of new virus particles is initially under the control of the host cell's transcription factors. Transcription of the proviral DNA into RNA is initiated by viral sequences in the long terminal repeat (LTR) (Tong-Starken, S.E., Luciw, P.A., and Peterlin, B.M. Human Immunodeficiency Virus Long Terminal Repeat Responds To T-cell Activation Signals. Proc. Natl. Acad. Sci. 1987;84:6845-6849). A certain number of the RNA molecules are used as genetic material while others are used as mRNAs to be translated into new viral proteins. The env proteins are postranslationally processed in the cell's Golgi apparatus and are transported into the host's cell membrane. Proteins that will be used for the core structure of the virus contain a fatty acid and these attach to the inside of the cell membrane. As all the components for the new virus accumulate, they bind to one another and form a spherical structure that bulges outward from the cell membrane. Two RNA molecules are placed into the developing virus particle. Lastly, the core associated enzymes (RT, integrase and protease) are postranslationally processed and the protease cleaves the core precursor proteins. The viral core proteins surround the viral RNA genome, the nearly completed virus encloses itself with a portion of the host cell membrane, and eventually the virus buds from the cell and is released. Infection by HIV, or other lentiviruses, is persistent and is usually characterized by a continuous, although relatively low level of virus production. A progressive increase in productive viral replication occurs and probably contributes to disease progression. This has led investigators to look for biological markers that may be associated with HIV-1 disease progression. Prognostic and Serological Markers of HIV-1 Progression Recently, many studies have focused on identifying specific biological markers associated with the progression of disease in HIV-1 infected individuals. The identification of markers that correlate with the progression of HiV-1 infection is vital for determining and understanding the pathogenesis of the disease. In addition, identification of markers that correlate with disease progression would aid in the development and monitoring of therapeutic agents. Early studies of HIV-1 pathogenesis found that the main target cell of HIV-1 was CD4+ T cells and that a significant decrease in their total number occurred as the disease progressed (Schittmann, S.M., Pallidopoulous, M.C., Lane, H.C., Thompson, L., Baseler, M., Massari, F., Fox, C.H., Salzman, N.P., and Fauci, A.S. The Reservoir For HIV-1 In Human Peripheral Blood Is A T cell That Maintains Expression Of CD4. Science 1989;245: 305-308; and .Klatzmann, D., Champagne, E., Chamaret, S. T-lymphocyte T4 Molecule Behaves As The Receptor For Human Retrovirus LAV. Nature 1986;234:1120-1123). Other studies investigated possible serum markers which could also be associated with disease progression and which either correspond to immune cell activation or reflect increased viral production. These markers included beta2 microglobulin, neopterin, and p24 antigenemia (Melmed, R.N., Taylor, J.M., Detels, R., Bozorgmehri, M., and Fahey, J.L. Serum Neopterin Changes In HIV Infected Subjects: Indicator Of Significant Pathology, CD4 T- Cell Changes, And The Development Of AIDS. J. Acquired Immune Deficiency Syndrome 1989;2:70-76). Beta2 microglobulin is part of the histocompatability complex (HLA) and is released from a T cell during immune activation and cell turnover. Normal levels measured in healthy individuals are less than 1.9 μg/μϊ (Hofmann, B., Wang, Y., Cumberland, W.G., Detels, R., Bozorgmehri, M., and Fahey, J.L. Serum Beta2- microglobulin Level Increases In HIV Infection: Relation To Seroconversion, CD4 T-cell Fall And Prognosis. AIDS 1990;4:207-214). Neopterin is a product of macrophage activation when these cells are stimulated by gamma interferon and reflects immune activation. Normal levels measured in healthy individuals are 6.62 nmol/L or lower (Fuchs, D., Hausen, A., Reibnegger, G., Werner, E.R., Dierich, M.P., and Wachter, H. Neopterin As A Marker For Activated Cell-Mediated Immunity: Application In HIV Infection. Immuno. Today 1988: 9:150-154). An increase above the normal levels of each marker reflects both lymphocyte and macrophage activation. Positivity for HIV-1 p24 reflects increased viral activity and production, and has been shown to be associated with poor prognosis (Allain, J.P., Laurian, Y., Paul, D.A., Verroust, F., Leuther, M., Gazengel, C., Senn D., Larrieu, M.J., and Bosser, C. Long-Term Evaluation Of HIV Antigen And Antibodies To p24 And gp41 In Patients With Hemophilia. - Enαl. J. Med. 1987;317:1 1 14-1 121 ) . Overall, the decline of CD4+ lymphocytes, as expressed as absolute numbers, was found to be the best predictor of HIV-1 progression. This was followed by the levels of neopterin or beta microglobulin, and finally the p24 antigen (Fahey, J.L., Taylor, L.M., Detels, R., Hofmann, B., Melmed, R., Nishanian, P., and Giorgi, J.V. The Prognostic Value Of Cellular And Serological Markers In Infection With Human Immunodeficiency Virus Type 1. N. En . J. Med. 1990;322:166-172). More recently, an increase in cellular and plasma viral load has been shown to be associated with clinical manifestations of HIV-1 disease and to correlate with a decrease in CD4+ cell count (Venet, A., Lu, W., Beldjord, K., and Andrieu, J.M. Correlation Between CD4 Cell Count And Cellular and Plasma Viral Load In HIV-1 Seropositive Individuals. AIDS 1991;5: 283-288). Culture techniques, requiring either isolated peripheral blood mononuclear cells (PBMC) or plasma from an infected individual, were used to determine the viral load (Ho, D.D., Moudgil, T., and Alam, M. Qύantitation Of Human Immunodeficiency Virus Type 1 In The Blood Of Infected Persons. - Engl. J. Med. 1989;321:1621 - 1625; Coombs, R.W., Collier, A.C., Allain, J.P., Nikora, B., Leuther, M., Gjerset, G.F., and Corey, L. Plasma Viremia In Human Immunodeficiency Virus Infection. N. Engl. J. Med. 1989;321:1626-1631). However, using this approach proved to be quite labor intensive and required a significant amount of time to complete. Later, methods were used to directly isolate viral particles or RNA from plasma either by using centrifugation techniques or direct extraction of viral RNA. The measurement of viral load was accomplished by reverse transcribing the viral RNA into a cDNA copy and amplifying the cDNA using the Polymerase Chain Reaction (PCR) (Bagnarelli, P., Memzo, S., Manzin, A., Giacca, M., Emanuele, V., and Clementi, M. Detection of Human Immunodeficiency Virus Type 1 Genomic RNA In Plasma Samples By Reverse Transcription Polymerase Chain Reaction. J. Med. Virology 1991 ;34:89-95). This resulted in a substantial reduction in assay time and a significant increase in sensitivity. The measurement of HIV-1 viral load in plasma has significant implications in monitoring disease progression and the efficacy of therapeutics. The direct measurement of plasma viral load indicates the level of active viral replication. The monitoring of viral replication, in combination with other markers for disease progression, may give a more precise indication of the pathogenesis of HIV-1 and may be a better predictor of disease progression. It may even serve as a means of determining the best time to implement therapy in seropositive individuals. SUMMARY OF THE PRESENT INVENTION Plasma HIV-1 viremia was monitored by immunocapture-cDNA/PCR in which the reverse transcription and amplification steps were carried out in a single tube. The direct lysis buffer of the present invention was formulated to isolate plasma HIV-1 RNA for direct use in the RT and PCR reactions without inhibiting enzymatic reactions, thus eliminating the need for organic solvent extraction and ethanol precipitation normally required to isolate nucleic acids. This resulted in a significant saving of time needed to complete the assay (saving approximately 16 hours) and may have decreased the likelihood of contamination due to decreased handling steps. A viral capture assay involving latex microparticles (0.1 μm) coated with monoclonal antibodies directed to the gp41 and gp120 envelope proteins of HIV-1 was used to capture cell free virions from serum/plasma. The standard parameters of the assay require that the plasma sample be incubated in the presence of the microparticles for three hours. In this study, the time was varied in order to determine whether the incubation time can be reduced without decreasing the sensitivity of the assay. The conventional method of extraction and purification of HIV-1 RNA from viral proteins requires several hours, excluding a final overnight ethanol precipitation. The method also requires multiple tube changes, which makes it relatively prone to contamination. To eliminate the organic solvent extraction and ethanol precipitation procedures, the present invention involves a series of buffers and conditions for the direct lysis of HIV-1 virions bound to the particles. Compatibility of the direct lysis buffer components with the reverse transcription and PCR enzymes is a major concern and particular attention was devoted to the formulation of a buffer that met this requirement. Lysis buffers included a single detergent at various concentrations. Ionic and non-ionic detergents were investigated as components of the direct lysis buffer. Also, the effectiveness of adding low concentrations of Proteinase K in combination with the various detergents was studied. Once the formulation of a direct lysis buffer was determined, the time and temperature conditions required for disruption of the viral membranes was determined. The standard method used to reverse transcribe the HIV- 1 RNA into a cDNA copy and its amplification by PCR requires two separate procedures. Maximum sensitivity was obtained by optimizing the assay components used in the two procedures (i.e., concentration of buffer, salt, primers, dNTPs and enzymes). However, there could possibly be a significant gain in assay sensitivity and time by combining the two procedures. A series of experiments was done to determine whether the reverse transcription and amplification procedures could be combined into one procedure. Compatibility of the direct lysis buffer with the RT and PCR assay components was maintained. The assay sensitivity was maintained by optimizing the MgC and dNTP concentrations, and other components if necessary. TABLE OF CONTENTS I. Background 1 Historical Background 1 HIV-1 Genetic Structure and Replication 2 Prognostic and Serological Markers for HIV-1 Progression 4 II. Summary 7 Brief Description of the Figures 9 III. Detailed Description 1 0 Plasma Viral Load in HIV-1 Infected Pregnant Women 1 1 Plasma Viral Load in HIV-1 Infected Blood Donors 1 1 II. MATERIALS AND METHODS 1 1 Reverse Transcription Controls 1 1 Amplification Controls 1 2 Immunocapture Controls 1 _ Inter- and Intrassay Contamination Control 1 3 Primer and Probe Preparation 1 3 Particle Preparation 1 3 Labeling of SK19 Probe 1 4 Viral Capture 1 5 Reverse Transcription 1 5 Polymerase Chain Reaction 1 6 Liquid Hybridization and Autoradiography. 1 6 Quantitation of Autoradiograph 1 6 III. RESULTS 1 7 Assay Control Characterization 1 7 Direct Lysis Buffer 1 7 Direct Lysis Buffer Incubation Time and Temperature 20 Viral Capture Time 20 Single Addition RT/PCR 20 Detection of Plasma HIV-1 RNA in Seropositive Pregnant Women 23 Detection of HIV-1 RNA in Seropositive Blood Donors 2 6 IV. DISCUSSION 26 V. BIBLIOGRAPHY 36 VI. CLAIMS 42 BRIEF DESCRIPTION OF THE FIGURES Figure 1 Assay Controls. A). Immunocapture controls, B). Reverse transcription controls, and C). Amplification controls. Autoradiography was done for three hours at -80°C. (Neg, Rn, and Dn represent negative controls for capture, RNA, and DNA, respectively.) Figure 2 Effect of various detergent concentrations on sensitivity of detection. Each detergent concentration was evaluated using the RNA controls R-5 and R-6, respectively. The standard RT/PCR procedures were used in the evaluation. The RNA controls were processed using the standard extraction procedure. Autoradiography was done for three hours at -80°C. Figure 3 Effect of direct lysis buffer on immunocapture controls. A). Organic solvent extraction. Direct lysis buffer containing B). 0.005% Triton X-100, C). 0.0045% Tween 20, D). 0.001 % SDS. Proteinase K was included as indicated. The standard RT/PCR procedure was used. Autoradiography was done for three hours at -80°C. Figure 4 Optimization of direct lysis time and temperature. Plasma controls were evaluated using the direct lysis buffer and the standard RT/PCR procedures. Autoradiography was done for three hours at -80°C. Figure 5 Optimization of viral capture time. Capture times were determined using the plasma controls and evaluated using the direct lysis buffer and standard RT/PCR procedures. Autoradiography was done for four hours at -80°C. Figure 6 Effect Of MgCl2 concentration on the single step RT/PCR procedure at a dNTP concentration of 50 mM. The controls were assayed using the standard RT/PCR procedure. The MgC concentrations ranged from 1.5 to 2.0 mM. Autoradiography was done for three hours at -80°C. Figure 7 Effect of MgCl2 concentration on the single step RT/PCR procedure at a dNTP concentration of 100 μM. The controls were assayed using the standard RT/PCR procedure. The MgC-2 concentrations ranged from 1.5 to 2.0 mM. Autoradiography was done for three hours at -80°C. Figure 8 Effect of MgCl2 concentration on the single step RT/PCR procedure at a dNTP concentration of 200 μM. A). Reverse transcription, B). Amplification, C). Immunocapture. The controls were assayed using the standard RT/PCR procedure. The MgCl2 concentration tested was 1.75 mM. Autoradiography was done for three hours at -80°C. Figure 9 Effect of assay volume on single step RT/PCR procedure. A). 50 μl procedure. B). 100 μl procedure. Autoradiography was done for three hours at -80°C. DETAILED DESCRIPTION OF THE INVENTION Determining the factors that relate to disease transmission and progression is a major concern in HIV-1 research. Recently, several studies have focused on identifying factors that may correspond to the transmission of HIV-1. In this regard, the correlation of HIV-1 viral load with transmission may be important. Two of the types of HIV-1 transmission include that from mother to child (vertical), and from blood donor to recipient (horizontal). Although the number of infections through vertical transmission of HIV-1 from a mother to her child is relatively low in the U.S., it does account for approximately 2% of new infections per year. Similarly, the current number of transfusion-associated HIV-1 transmissions is extremely low. However, determining whether there is a correlation between HIV-1 viral load and transmission of HIV-1 in these two cases will increase the understanding of the pathogenicity of HIV- 1 . Plasma Viral Load in H1V-1 Infected Pregnant Women Samples were collected from transmitting and non-transmitting pregnant women, determined retrospectively by the loss or persistence of H1V-1 antibodies after 15 months in the infant (provided by Case Western Reserve University and the University of Washington, Seattle). Duplicate viral capture and RT/PCR for each sample were performed on coded samples. A consensus semi-quantitative value (3+, 2+, 1+, negative) was assessed. The semi-quantitative plasma RNA viral load was compared to other possible virologic markers of transmission (p24 antigenemia, CD4+ count and beta2 microglobulin levels) to determine its clinical usefulness for the prediction of ' • HIV-1 vertical transmission. Plasma Viral Load in HIV-1 Infected Blood Donors Plasma samples from HIV-1 seropositive blood donors were obtained (Transfusion Safety Study Repository, San Francisco, CA), and HIV-1 plasma viral load was determined retrospectively. Samples were selected from a pool of 78 samples known to have infected the recipient and 12 that did not infect the recipient. A total of twenty two samples were tested, with an equal number of samples coming from those that did infect and those that did not infect the recipient. Duplicate viral capture and RT/PCR for each sample were performed. A consensus semi-quantitative value was assessed. The semi- quantitative plasma RNA viral load was compared to other virologic markers of transmission (p24 antigenemia, CD4+ count and beta2 microglobulin) to determine the association of HIV-1 viral load in transfusion-associated transmission of HIV-1. MATERIALS AND METHODS Preparation of Controls Reverse Transcription Controls. To monitor the efficiency of the reverse transcription procedure, a set of calibrated RNA samples was prepared from the HIV-1 NIB chronically infected H9 cell line (Abbott Laboratories). Total cellular nucleic acids were extracted with guanidinium thiocyanate, and the RNA was purified by centrifugation through a cesium chloride (CsCI) cushion (Chirgwin, J. M., Prsybla, G., MacDonald, P.J., and Rutter, W. J. Isolation Of Total Cellular RNA. Biochem. 1979;18:5294-5299). The RNA pellet was dissolved to 0.5 μg/ml with dd^O and serially diluted 10 fold into ddH 0 containing 20 μg/ml yeast tRNA (GIBCO-BRL, Gaithersburg, MD). Samples diluted 10 5 and 10 6 fold, referred to as R *5 and R-* 3 , were the two lowest dilutions that consistently gave positive results after reverse transcription and amplification. These samples were used as controls. Amplification Controls. The HIV-1 LAV infected cell line 8E5 (Memorial Sloan Kettering Institute, New York), which contains one copy of proviral HIV-1 per cell, was used to prepare amplification controls. Total genomic DNA from 10 6 cells, representing 10 6 HIV-1 copies, was extracted for one hour at 56°C in 500 μl of a solution containing 10 mM TRIS, pH 8.3, 0.5 mg/ml Proteinase K (Promega, Madison, Wl) and 0.25% SDS (Sigma, St. Louis, MO). The lysate was then extracted with an equal volume of phenol (pH 7.0), then with chloroform (adjusted to 0.3 M NaOAc) and was then precipitated with twice the volume of absolute ethanol at -20°C for 16 hours. The DNA was centrifuged (Hill Scientific mv13) at 10,000 rpm for 10 minutes, the supernatant removed, and the pellet washed with ice cold 70% ethanol. The DNA pellet was dissolved in 0.5 ml 10 mM TRIS, pH 8.0, 100 mM NaCI. The purified DNA, corresponding to approximately 10 6 HIV-1 provirus copies, was serially diluted 10 fold into dd^O containing 20 μg/ml salmon sperm DNA (Sigma, St. Louis, MO). Fifty microliters of a 10 "3 and 10 "4 dilution (corresponding to 100 and 10 HIV-1 proviral copies, respectively) were used as amplification controls. Immunocapture Controls. To verify that the viral capture procedure gave reproducible results, a set of calibrated plasma controls was prepared. Supernatant from H9 cells chronically infected with HIV-1 NIB (Abbott Laboratories) was obtained and serially diluted 10 fold in seronegative plasma. The dilutions were tested by viral capture followed by RT/PCR, and the two lowest positive dilutions (referred to as MK" 4 and MK "5 ) were used as positive controls. Inter- and Intra-assav Contamination Control The most common cause of false positive results in a PCR procedure is carryover of previously amplified DNA, while sample to sample contamination also contributes to the problem. To minimize the possibility of contamination during sample handling and the PCR procedure, a series of steps were routinely followed. Plastic disposable, single use beakers were used to prepare reagents. Bottled distilled water, free of any RNase (Abbott Laboratory, Catalog #NDC 0074-7139-09) was used to prepare all reagents. All reagents were aliquoted into single use tubes and were stored at -20°C until used. Sample handling, amplification, and detection were done in three separate laboratories. Sample handling was done in a laminar flow hood. Latex gloves were always worn and were changed numerous times during the assay. Each laboratory contained a separate set of pipetmen, and barrier pipet tips were used throughout the procedure. Amplification was done in an acrylic biosafety cabinet. The biosafety cabinet, as well as the PCR laboratory, was equipped with a UV light source, and UV sterilization (American Ultraviolet Company, Murry Hills, NJ) was performed weekly in order to control for any possible RNA/DNA contamination. Negative controls for the immunocapture, reverse transcription and the PCR were included with each assay. An assay was considered invalid if any one of the three negative controls gave a positive result. Primer and Probe Oligonucleotide Preparation The primers SK38/SK39 (representing nucleotides 1551-1578 and 1638- 1665, respectively) and the probe SK19, representing nucleotides 1597-1635 of the HIV-1 (HIVSF2, Genebank K02007) gag region were synthesized at Abbott Laboratories using an Applied Biosciences, Inc. 380 Synthesizer (Foster City, CA) and HPLC purified with a Waters Photodioarray 990 (Milford, MA). Particle Preparation Carboxylated latex microparticles (0.1-0.3 μm diameter, Seradyn Inc., Indianapolis, IN) were covalently coupled with HIV-1 monoclonal anti-gp120 and anti- gp41 IgG (Abbott Laboratories) using 1 -ethyl-3,3-(dimethyl aminopropyl) carbodiimide chemistry (EDC) (Sondergard-Anderson, J., Lauritzen, E., Lind, K., and Holm, A. Covalently Linked Peptides For Enzyme-Linked Immunosorbent Assay. J. Immuno. Methods 1990;131:99-104). After coupling, the microparticles were centrifuged for 30 minutes at 17,000 x g (Beckman J2-21M), and the supernatant was discarded. The microparticles were washed two times with equal volumes of wash buffer (PBS containing 2% Tween 20) and were then resuspended to volume with overcoat buffer (150 mM TRIS, pH 8.0, 100 mM NaCI, 0.5% porkskin gelatin, 0.1% Tween 20, 9.5% sucrose and 0.02% NaNβ). After incubating in overcoat buffer for 16 hours at 45°C, the microparticles were pelleted, and the supernatant was discarded. The microparticles were resuspended to 50% of their original volume with storage buffer (65.5 mM TRIS, 84.5 mM TRIS HCI, pH 8.0, 100 mM NaCI, 0.4 M sucrose, 1% porcine skin gelatin, and 0.1% Tween 20), and the percentage of solid was determined by comparing the A500 of a diluted fraction to a standard curve of known solids. The microparticles were adjusted to a predetermined percent solids with storage buffer and were stored at 2-8°C until used. Labeling pf SK19 Probe The SK19 oligonucleotide (5'-ATCCTGGGATTAAATAAAATAGAA GAATGTATAGCCCTAC) was labeled with 32 Pθ4 at the 5' terminus by using T4 polynucleotide kinase. The reaction consisted of 5.0 mM TRIS HCI, pH 8.0, 1.0 mM MgCI 2 , 5.0 mM NaCI, 1.0 μg SK19, 50 uCi gamma 32 P ATP (Amersham, 3000 Ci/mmol) and 10 Units of T4 kinase (New England BioLabs, Beverly, MA), in a total volume of 10 μl. The reaction was carried out for 30 minutes at 37°C, followed by inactivation of the T4 kinase for 5 minutes at 95°C. To separate the labeled probe from unincorporated *- *2 P ATP, the reaction mixture was electrophoresed through a 10% polyacrylamide gel (29.25 ml H2O, 2.25 ml 10x TBE [Sondergard-Anderson, J., Lauritzen, E., Lind, K., and Holm, A. Covalently Linked Peptides For Enzyme-Linked Immunosorbent Assay. J. Immuno. Methods 1990;131 :99-104], 11.5 ml polyacrlyamide:bis (19:1), 30 μl 10% ammonium persulfate, and 30 μl TEMED) for one hour at 200 volts. The DNA was stained with 0.5 μg/ml ethidium bromide in ddH2θ for five minutes and was visualized using long wavelength UV illumination (LKB 2011 Macrovue). The band representing the labeled probe was excised and placed into a 1.5 ml eppendorf tube containing 0.5 ml STE (100 mM NaCI, 10 mM TRIS, pH 8.0 and 1 mM EDTA). To elute the probe from the gel fragment, the tube was rotated (Labquake Shaker, Berkley, CA) for 16 hours at room temperature. Three microliters of eluted probe, corresponding to approximately 6.0 ng, was used to determine the labeling efficiency. The specific activity of the labeled SK19 probe was routinely between . 1x10 7 and 2x10 7 cpm/μg. The labeled probe was stored at -20°C until used. Viral Capture Immunocapture of HIV-1 virions was carried out in a 1.5 milliliter Eppendorf tube containing phosphate buffered saline (150 μl; PBS, 137 mM NaCI, 2.68 mM KCI, 12 mM Na2HP0 , 1.76 mM KH2PO4), anti-HIV-1 antibody coated microparticles (50 μl) and plasma (50 μl). The mixture was incubated for three hours at room temperature on a rocking platform (20 rpm, Thermolyne VariMix), then centrifuged for 10 minutes at 5000 rpm, and the supernatant was either saved or discarded. Genomic HIV-1 RNA was extracted by the addition of a Proteinase K SDS solution (200 μl; 10 M TRIS, pH 7.4, 0.25% SDS, 0.5 mg/ml Proteinase K, and 10 μg/ml yeast tRNA) and further incubation (one hour at 56°C). The HIV-1 RNA was purified by extraction with an equal volume of phenol, followed by extraction with an equal volume of chloroform (adjusted to 0.3 Ivf NaAOc) and was precipitated with twice the volume of absolute ethanol at -20°C for 16 hours. The RNA was pelleted by centrifugation (Hill Scientific mv13) for 10 minutes at 12,000 rpm, and the supernatant was discarded. The RNA pellet was washed with ice cold 70% ethanol, centrifuged as before, the supernatant discarded, and the RNA dissolved in 30 μl of dd^O). Reverse Transcription Viral RNA was reverse transcribed into cDNA using the enzyme reverse transcriptase, from Avian Myeloblastosis Virus (AMV-RT, Gibco-BRL). Duplicate aliquots (15 μl) of isolated HIV-1 genomic RNA were placed into 0.5 ml centrifuge tubes containing 5.0 μl of RT mix. The final RT reaction contained 10 mM TRIS, pH 8.3, 2 mM MgCI 2 , 50 mM KCI, 20 mM DTT, 0.001% gelatin, 25 μM each dNTP (Pharmacia, Piscataway, NJ) and 25 ng of SK38/SK39 primers, (SK38 5'- ATAATCCACCTATCCCAGTAGGAGAAAT, SK395'-TTTGGTCCTTGTCTTATGTCCAGAATGC). The tubes were heated to 95°C for five minutes, centrifuged, and reverse transcriptase (2.0 U; Gibco-BRL) containing 8.0 U of RNasin (Promega, Madison, Wl) was added. The RT reaction was carried out for 30 minutes at 42°C. Thirty microliters of water and two drops of mineral oil (Sigma, St. Louis, MO) were then added, and the reverse transcriptase was heat inactivated at 95°C for five minutes. Polymerase Chain Reaction Amplification of HIV-1 DNA proceeded by addition of 50 μl of PCR mix to each sample. The final PCR reaction contained 10 mM TRIS, pH 8.3, 1.5 mM MgC.2, 50 mM KCI, 0.001% gelatin, 50 μM each dNTP, 50 ng SK38/39 primers and 1.0 U Taq Polymerase (Cetus, Norwalk, CT). Included in each run were a set of DNA controls representing known amounts of HIV-1 provirus to which the assay could be compared and thus quantitated. The amplifications were performed with a Perkin Elmer Cetus model 480 Thermocycler programmed for 35 cycles of denaturation at 94°C for one minute and annealing/extension at 56°C for two minutes. Detection of Amplified Fragment Liquid Hybridization and Autoradiography. Amplified HIV-1 DNA was detected by hybridization of 5 μl of 32 P SK 19 probe (2.5 μl probe plus 2.5 μl R3 buffer [50 mM TRIS HCI, pH 8.0, 10 mM Mgbl 2 , 100 mM NaCI]) (1-2x10 7 cpm/μg) to 15 μl of amplified material. The amplified material and probe were mixed and heated for 10 minutes at 100°C (to separate the double stranded amplified fragments), centrifuged to return the condensation to the bottom of the tube, and allowed to anneal for 30 minutes at 56°C. Five microliters of loading dye (0.25% bromophenyl blue, 40% sucrose) was added to each sample, and the entire volume was loaded onto a 10% polyacrylamide gel. Eiectrophoresis was done at 150 V for 30 minutes, followed by 250 V for 1.5 hours. The gel was placed onto a piece of Whatman blot paper, covered with plastic wrap, and autoradiographed for one and four hours at -80°C using an intensifying screen (DuPont Cronex). Development of the autoradiograph was done with a Kodak M35A X-OMAT Processor. Quantitation of Autoradiograph. Results were quantitated by directly comparing the sample results to the plasma and known DNA copy number controls. In some situations, exact comparisons of densitometer readings were used to quantitate the results. RESULTS Assay Control Characterization The efficiency of the immunocapture, reverse transcription and amplification procedures was monitored by using specific controls. Immunocapture controls consisted of the two lowest positive serial dilutions of a tissue culture supernatant from an HIV-1 IIIB infected H9 cell line. Reverse transcription controls consisted of the two lowest positive serial dilutions of a purified preparation of RNA extracted from HIV-1 IIIB infected H9 cell line. Amplification controls consisted of purified DNA obtained from the HIV-1 LAV infected 8E5 cell line, which contains one copy of HIV-1 per cell. The specific amplification products produced by the three types of controls are shown in Figure 1. Usually, the detection of each set of controls was consistent between assays, and verified the sensitivity of the assay. In addition, negative controls for the immunocapture, reverse transcription, and amplification procedures were included in each assay to verify the specificity. Optimization of Assay Parameters The viral capture assay uses latex microparticles (0.1 μm) covalently coupled with monoclonal antibodies directed to the gp41 and gp120 envelope proteins of HIV-1 , which capture cell free virions from serum/plasma. The original protocol for immunocapture included a three hour incubation, followed by Proteinase K/SDS digestion, a phenol-chloroform extraction, an ethanol precipitation to purify the viral RNA, reverse transcription, and amplification by the polymerase chain reaction. The total time required to complete this part of the assay was five to seven hours. Additionally, detection of the amplified material required liquid hybridization, gel electrophoresis, and autoradiography. Because many of the steps in this protocol were determined empirically, improvements in some or all of the steps prior to detection were sought to simplify the overall procedure and decrease the total time requirement, and the probability, of contamination. Direct Lysis Buffer. Purification of viral RNA by phenol/chloroform extraction and ethanol precipitation is labor intensive and time consuming. However, this purification is conventionally performed in order to overcome the inhibitory effect of SDS on the polymerase chain reaction and to- remove excess Proteinase K which may interfere with the assay (Erlich, H.A. PCR Technolo gy . Principles and Applications fpr DNA Amplification. Stockton Press, 1989. New York, NY. pp. 17-22). To eliminate the use of organic solvents and the need for ethanol precipitation, a series of direct lysis buffers were examined. The objective was to formulate a buffer that would be compatible with the reverse transcription and amplification procedures, while yielding results comparable to the standard digestion/extraction procedure. The most common reagents used to disrupt cellular and viral membranes include the ionic detergent sodium dodecyl sulfate (SDS), and the non-ionic detergents Triton X- 100 and Tween 20. Proteinase K was used in combination with these detergents in order to digest proteins which may be associated with nucleic acids. The Triton X-100 and Tween 20 concentrations ranged from 0.1-0.5%. These two detergents, when used at concentrations of 0.5% or lower, do not interfere with the Taq polymerase during the amplification procedure. SDS on the other hand, inhibits the polymerase chain reaction 99% and 90% when present in concentrations of 0.1% and 0.01%, respectively. SDS does not inhibit the Taq polymerase when used at concentrations of 0.001% or lower (Erlich, H.A., above). The components used in the standard lysis of the viral membranes included 0.25% SDS and 500 μg/ml Proteinase K. However, as mentioned previously, the concentrations needed to lyse the viral membrane in a direct lysis buffer and also be compatible in the reverse transcription/amplification procedures may need to be significantly reduced. With this in mind, lysis buffers containing low concentrations of Triton X-100, Tween 20 or SDS were evaluated. In addition, a low concentration of Proteinase K was added to the lysis buffers containing low concentrations of detergent. In this case, the lysis buffer was heated at 95°C for 10 minutes to inactivate the Proteinase K prior to initiating the RT and PCR procedures. To provide a buffering system, 10 mM TRIS, pH 7.0, was chosen and was included in all lysis buffers examined. The composition of the various direct lysis buffers which were examined are presented in Table 1. The standard assay using the RNA and DNA controls was initially used to evaluate the lysis buffers at an incubation time and temperature of 56°C for one hour. Table 1. Composition of the Lysis Buffers Investigated Buffer 1 TRIS (mM) Triton X-100 (%. 1 .1 1 0 0.5 1 .2 1 0 0.05 1.3 1 0 0.005 Buffer 2 TRIS (mM. Tween 20 .%. 2.1 1 0 0.45 2.2 1 0 0.045 2.3 1 0 0.0045 Buffer 3 TRIS (mM. Proteinase K SDS (%) (μg/m l) 3.1 1 0 NA 0.1 3.2 1 0 NA 0.01 3.3 1 0 NA 0.001 Buffer 4 4.1 1 0 1 .0 0.1 4.2 1 0 1 .0 0.01 4.3 1 0 1 .0 0.001 The buffers containing 10 mM TRIS and various concentrations of Triton X-100 produced signals similar to those obtained with the standard procedure. Similarly, buffers containing various concentrations of Tween 20 produced signals similar to those obtained with the standard procedure. Of the three lysis buffers containing 10 mM TRIS and various concentrations of SDS, only the one containing 0.001% gave results similar to the conventional procedure. The lysis buffers containing 0.1% and 0.01% SDS generated no signals. This confirmed the inhibitory effect that SDS has on the Taq polymerase. Similarly, lysis buffers containing SDS and 1 μg/ml of Proteinase K also showed a decrease in sensitivity as the SDS concentration increased above 0.001%. Only the lysis buffer containing 0.001% SDS gave results comparable to the standard method (Figure 2). The efficiency of the various lysis buffers to disrupt the HIV-1 virions and their effect on the RT/PCR reactions in the presence of the immunoparticles was determined using the immunocapture controls. The analysis was limited to the use of the lowest detergent concentrations in order to minimize any possible adverse effect on the enzyme reactions. As shown in Figure 3, direct lysis buffers containing either of the three detergents and one μg/ml Proteinase K resulted in signals identical to the control. The removal of Proteinase K from the direct lysis buffers resulted in significantly reduced signals. The use of either of the three detergents resulted in the efficient lysis of the HIV- 1 virions, and none of the three detergents appeared to affect the enzyme reactions. An exemplary lysis buffer containing 10 mM TRIS, pH 7.0, 0.001% SDS, and 1.0 μg/ml Proteinase K was used in the assays. In summary, direct lysis with the buffer containing 10 mM TRIS, pH 7.0, 0.001% SDS and one μg/ml Proteinase K gave comparable results to the standard digestion buffer containing 0.25% SDS and 500 μg/ml Proteinase K (Figure 3 vs 5D). Unlike the conventional digestion buffer, this formulation involved a two log decrease in overall concentrations of SDS and Proteinase K and the elimination of the yeast tRNA. Direct Lysis Buffer Incubation Time and Temperature. The minimum time and •I temperature required to disrupt the viral membrane and release the genomic RNA from the core proteins was determined using the direct lysis buffer. The parameters of the standard procedure, 56°C for one hour, were used as control. Two conditions were tested: incubation for 30 minutes at 56°C and incubation for 30 minutes at 37°C. The combined effects of time and temperature on assay sensitivity using the direct lysis buffer are shown in Figure 4. The sensitivity of RNA detection using direct lysis buffer for 30 minutes at 37°C was identical to that of the standard procedure. The slight differences in band intensity are a common occurrence and reflect the inter-assay variability of the enzymatic reactions. Thus, these parameters were chosen for all subsequent assays. Viral Capture Time. The minimal time required for immunocapture of the plasma-associated virions by the anti-HIV-1 antibody-coated microparticles was determined. The effect of reducing the capture time from three hours to two hours and one hour at ambient temperature was evaluated. The results obtained after capture for one hour were equivalent to those obtained with the standard procedure (Figure 5). Therefore, all subsequent assays were done using immunocapture for one hour. Single Addition RT/PCR Buffer. In the standard reverse transcription procedure, 15 μl of sample was added to 5 μl of RT buffer. After heat denaturation, one microliter of Reverse Transcriptase/RNasin was added, and reverse transcription was carried out for 30 minutes at 42°C. Afterward, 30 μl of ddH 2 0 and two drops of mineral oil were added and the mixture was heated at 95°C for five minutes. Taq mixture, containing all the components for amplification, was added, and amplification was initiated. These multiple additions, a total of six, to each assay tube made the whole procedure cumbersome and increased the risk of contamination by carryover from adjacent tubes. Recently, a method has been described where RT/PCR of genomic HCV RNA was done in a single tube using a single addition of buffer and enzymes (Lin, H.J., Naiyi, S., Mizokami, M., and Hollinger, F.B. Polymerase Chain Reaction Assay For Hepatitis C Virus RNA Using A Single Tube For Reverse Transcription And Serial Rounds Of Amplification With Nested Primer Pairs. J. of Med. Virology 1992;38:220-225). To determine whether this type of process could be used to reverse transcribe and amplify HIV-1 viral RNA, several single addition RT/PCR procedures were tested. The final volume for the HIV-1 single addition RT/PCR procedure was chosen to be 100 μl, which was the volume the •I standard method used. Since the assay used 15 μl of sample, an additional 85 μl, which contained all the necessary components for the RT and PCR procedures, had to be added. The same general assay format was used for the single step RT/PCR procedure. Following direct lysis, 15 μl of sample was placed into a 0.5 ml centrifuge tube, heated to 95° C for five minutes to denature the Proteinase K in the lysis buffer, centrifuged, and 85 μl of the amplification mixture was added, followed by two drops of mineral oil. Reverse transcription was carried out for 45 minutes at 42°C, followed by denaturation of the reverse transcriptase at 95°C for three minutes. Amplification was directly initiated using the standard parameters. Each sample was assayed in duplicate, and both RT and PCR procedures were done with a thermocycler. To achieve comparable sensitivity to that of the standard method, the single addition assay component concentrations had to be optimized. The TRIS, KCI, gelatin, and DTT concentrations were identical in both the standard RT and PCR procedures, and their concentrations were chosen for the single addition procedure. Therefore, they were not modified. The two most critical parameters affecting the sensitivity of the reverse transcription and amplification procedures are the MgCl2 and dNTP concentrations (Yong, W.H., Wyman, S., and Levy, J.A. Optimal Conditions For Synthesizing Complementary DNA In The HIV-1 Endogenous Reverse Transcriptase Reaction. AIDS 1990;4:199-206). The optimal MgCI 2 concentrations for the standard RT and PCR procedures were 2.5 mM and 1.5 mM, respectively. The optimal dNTP concentrations for the standard RT and PCR procedures were 25 μM and 50 μM, respectively. Since both the RT and PCR procedures were to be done with a single addition of reagents, these two components concentrations had to be reoptimized. The MgC*2 concentrations used to determine optimal sensitivity were 1.5, 1.75, and 2.0 mM. The dNTP concentrations tested were 50, 100 and 200 μM. At a dNTP concentration of 50 μM, and with varying concentrations of MgCl2, the overall signal intensity decreased significantly in the controls tested (Figure 6). Furthermore, the signal intensity decreased as the MgCtø concentration increased. At 2.0 M MgC.2, the RT and amplification controls produced weak but detectable signals. In contrast, there was no detectable signal generated for the immunocapture controls. Therefore, it appeared that a dNTP concentration of 50 μM was too low for optimal detection in the single step RT/PCR procedure. The concentration of dNTP was raised to 100 μM, and the same concentrations of MgCl2 were evaluated. As shown in Figure 7, there were slight decreases in RT and amplification control signals as the MgC.2 concentration increased. However, there did not appear to be the same significant decrease in amplification control signal as in the previous set of experiments. The decrease in RT and amplification control signals that occurred as the MgCl2 concentration increased and the dNTP concentration was at 100 μM indicates that the dNTP concentration may be somewhat rate limiting. An experiment was carried out by using the lowest optimal concentration of MgCl2 (1.75 mM) with an increased dNTP concentration (200 μM) to ensure that these concentrations would generate signals comparable to the standard procedure. As shown in Figure 8, the single step RT/PCR procedure (involving 1.75 mM MgCl2 and 200 μM dNTP) produced control signals similar to the standard procedure. To ensure that the dNTP concentration was not rate limiting their final concentration was chosen as 200 μM and the MgCl2 concentration was set at 1.75 mM. After optimizing the single step RT/PCR procedure in a final volume of 100 μl, an objective was to reduce the volume to 50 μl in order to save on reagent use. The 50 μl procedure gave excellent results when both the RNA and DNA controls were tested (Figure 9). However, no signal was obtained when plasma controls were tested. Three sets of plasma controls were actually evaluated. Of the six MK *4 reactions, three gave an extremely weak signal, and three were negative. None of the MK "5 controls gave a signal. Therefore, a final volume of 100 μl was chosen for the single step RT/PCR procedure. In summary, the final amplification mixture for the single step RT/PCR procedure contained 10 mM TRIS, pH 8.3, 50 mM KCI, 5 mM DTT, 0.001% gelatin, 1.75 mM MgCI 2 , 200 ng primers, 200 μM each dNTP, 10 U RNasin, 2.5 U AMV RT, and 1 U Taq Polymerase in a final volume of 100 μl. This amplification mixture provided results comparable to the standard methodology. The sensitivity was not affected, and the mixture actually appeared to produce slightly stronger control signals than the standard procedure. The final assay procedure was established as: a) viral capture for one hour at room temperature, b ) centrifuge at 5000 rpm for 10 minutes, discard supernatant, wash with 150 μl PBS, centrifuge as before and discard supernatant, c) add 30 μl of direct lysis buffer, vortex briefly and incubate for 30 minutes at 37°C, d) place duplicate 15 μl aliquots into separate 0.5 ml centrifuge tubes, and heat at 95°C for 5 minutes, e) add 85 μl amplification buffer, two drops of oil, reverse transcribe at 42°C for 45 minutes, heat denature and amplify. Detection of Plasma HIV-1 RNA in Seropositive Pregnant Women Although the factors affecting the vertical transmission of HIV-1 from an infected mother to her child are unknown, preliminary evidence suggests that viral load may have a significant role. The following was undertaken to determine whether a higher maternal HIV-1 plasma viral load correlated with an increased likelihood of vertical transmission. Coded plasma samples were obtained at the time of delivery from 49 seropositive pregnant women selected from studies initiated in the United States and Uganda, Africa. The plasma samples were selected from mothers known either to transmit or not transmit HIV-1 to their child. Overall, there were 21 women who did transmit HIV-1 to their children and 28 who did not. The U.S. cohort consisted of 4 transmitting and 16 non-transmitting mothers. The Ugandan cohort consisted of 17 women who transmitted and 12 who did not transmit HIV-1. Additionally, serological data available for the U.S. and Ugandan mothers consisted of CD4 count and beta2 microglobulin levels, respectively. At the time of delivery, all mothers were clinically asymptomatic. There was no significant association between the detection of HIV-1 RNA in maternal plasma and vertical transmission of HIV. Overall, 4 of the 22 transmitting mother samples tested were found to be HIV-1 RNA positive, while 1 1 of the 27 non- transmitting mother samples were HIV-1 RNA positive (Table 2). Table 2. Detection of Plasma HIV-1 RNA in Seropositive Women (Data given as RNA positive/total.) Transmitting Nontransmittinq U.S. 2/ 4 6/ 1 6 Uganda 2/ 1 7 5/ 1 2 Overall 4/22 * 1 1 /28 2 samples were from the same mother In the U.S. group (Table 3A), an equivalent proportion of transmitting and non- transmitting mothers were positive for HIV-1 RNA (40 and 38%, respectively). As expected, positivity for HIV-1 RNA correlated with lower CD4+ count. Women with detectable HIV-1 RNA had a median CD4+ count of 257/mm 3 (interquatrile range: 161-418/mm 3 ), while those having no detectable HIV-1 RNA had a median CD4+ count of 966/mm 3 (interquatrile range: 591 -1113/mm 3 ). However, four women who had fewer than 500 CD4+/mm 3 and had detectable HIV-1 RNA did not transmit the virus to their infants. Additionally, one woman having CD4+ count of 1113/mm 3 and no detectable plasma HIV-1 RNA transmitted the virus during two successive pregnancies. Among the Ugandan mothers (Table 3B), 12% who did transmit, compared to 42% of mothers who did not transmit, had detectable HIV-1 RNA. The beta2 microglobulin levels were not significantly different between the two groups, 1.66 vs 1.60 μg/μl (transmitting and non-transmitting, respectively). Table 3 Serology of U.S. and Ugandan Cohorts Serology of A) U.S. and B) Ugandan Mothers. CD4+ values are expressed as per mm3 and beta rnicroglobulin levels are expressed as μg/μl. ND = not determined. Detection of HIV-1 RNA in Seropositive Blood Donors HIV-1 viral load in the plasma of blood donors may play an important role in transfusion-associated transmission of HIV-1. To investigate this, HIV-1 viral load was measured in plasma samples obtained from the Transfusion Safety Study Repository (San Francisco, CA). Twenty two samples were selected from a pool of 78 infectious and 12 non-infectious samples. Of the 22 seropositive samples selected, there were 11 transfusion associated transmissions and 11 non-transmissions (Table 4). The CD4+ lymphocyte counts could not be determined for the original samples, but approximately one year after collection, the average CD4+ lymphocyte count for the transmitting and non-transmitting donors were 470 and 746 per μl, respectively. Table 4 Detection of Plasma HIV-1 RNA From Transmitting and Non-transmitting Blood Donors RNA + RNA - Transmitting 7 4 Non-transmitting 0 1 1 Overall, 7 of the 22 plasma samples were found to be HIV-1 RNA positive. Of the 11 infectious donations, 7 were HIV-1 RNA positive, while none of the 11 non- infectious donations were HIV-1 RNA positive. Therefore, there was a strong correlation between infectivity and the presence of HIV-1 RNA in the donor samples. DISCUSSION The detection of HIV-1 infection is a major factor in controlling the spread of the virus. Currently, antibody EIA and Western Blot tests are used to identify an HIV-1 infection. Recently, interest has focused on monitoring the progression of HIV infection and the response to therapy in HIV infected individuals. A number of methods, primarily based on p24 antigen detection, culture and nucleic acid amplification, have been developed to monitor HIV-1 disease progression. Quantitative cell and plasma culture can measure HIV-1 infectious titer from peripheral blood mononuclear cells (PBMC) and from plasma. Studies have shown that 2 6 the number of infected PBMC varied, depending on the clinical stage of the individual, and ranged from 1:50,000 in asymptomatic to 1:400 (infectedmormal) in AIDS individuals (Ho, D.D., Moudgil, T., and Alam, M. Quantitation Of Human Immunodeficiency Virus Type 1 In The Blood Of Infected Persons, ti. Engl. _\. Med. 1989;321:1621- 1625). Similarly, it has been shown that detection of cell-free virus in plasma also reflects the clinical stage of infection (Coombs, R.W., Collier, A.C., Allain, J.P., Nikora, B., Leuther, M., Gjerset, G.F., and Corey, L. Plasma Viremia In Human Immunodeficiency Virus Infection. N. Engl. J. Med. 1989;321 :1626-1631). However, both methods require special facilities and are significantly expensive and time consuming. In addition, cell culture requires the in vitro activation of the isolated cells, which may not represent an actual in vivo situation. Also, the sensitivity of plasma culture is not sufficient to detect a majority of known infections, and its reproducibility depends on the stimulated donor cells used in the assay (Eschaich, S., Ritter, J., Rougler, P., Lepot, D., Lamelin, J.P., Sepetjan, M., and Trepo, C. Plasma Viremia As A Marker Of Viral Replication In HIV Infected Individuals. AIDS 1991 ;5:1 189-1 194). The polymerase chain reaction (PCR) has been used to detect HIV-1 DNA present in infected PBMCs or virus associated HIV-1 RNA. Quantitative detection of proviral HIV-1 DNA isolated from PBMCs is very sensitive. However, it is not known whether all of the measured HIV-1 DNA corresponds to transcriptionally active DNA. Studies have shown that an increase in plasma HIV-1 RNA viral load is a good predictor of disease progression. Thus, the measurement of virus associated HIV-1 RNA by PCR should be extremely sensitive and useful. However, the isolation of the virus from plasma requires either ultracentrifugation or guanidine isothiocyanate extraction (Aoki-Sei, S., Yarchoan, R., Kageyama, S., Hoekzema, D.T., Pluda, J.M., Wyvill, K.M., Broder, S., and Mitsuya, H. Plasma HIV-1 Viremia In HIV-1 Infected Individuals Assessed By Polymerase Chain Reaction. AIDS Res, and Human Retro. 1992;8:1263-1270; Scadden, D.T., Wang, Z., and Groopman, J.E. Quantitation Of Plasma Human Immunodeficiency Virus Type 1 RNA By Competitive Polymerase Chain Reaction. J. Infect. Diseases 1992;165:1119-1123; Holodniy, M., Katzenstein, D.A., Sengupta, S., Wang, A.M., Casipit, C., Schwartz, D.H., Konrad, M., Groves, E., and Merigan, T.C. Detection And Quantification Of Human Immunodeficiency Virus RNA In Patient Serum By Use Of The Polymerase Chain Reaction. J. Infect. Diseases 1992;163:862-866). Like the culture techniques, these methods are labor intensive and require a great deal of time and expertise to complete. The present inventions is useful in an assay using immunocapture of plasma associated HIV-1 virions for the direct measurement of HIV-1 replication (viremia). This type of assay represents a sensitive and specific method for measurement of plasma associated HIV-1 virus without the need for culture techniques or cumbersome chemical extraction procedures. A direct lysis buffer was formulated that was used directly with a simplified method of reverse transcription and amplification of HIV-1 genomic RNA. These changes considerably reduced the time required to monitor HIV-1 viral load. A particle size of 0.1 to 0.3 μ was chosen because of a general increase in surface area obtained per unit volume. Theoretically, this would maximize the quantity of antibody coupled onto the particles and this should, in turn, increase the sensitivity of the immunocapture. High affinity monoclonal antibodies, rather than polyclonal antibodies, were used to capture the HIV-1 virions in order to maximize sensitivity. Both the gp120 and gp41 proteins are components of the virus outer membrane and are present on every infectious HIV-1 virus particle. Thus, anti-gp120 and anti-gp41 specific monoclonal antibodies were selected for immunocapture. The method of attachment of the antibody to the particle may affect both the sensitivity and specificity of the assay. There are two methods routinely used to attach antibodies to a solid matrix. The first, and probably the simplest, is passive adsorption, and the second is covalent attachment. The specific attachment of the antibody depends on the chemical composition of the particle. Passive adsorption is achieved by relying on ionic and hydrophobic interactions between the particle and antibody. Covalent attachment relies on production of a covalent bond between the particle and the antibody. Carboxylated particles were used for the covalent attachment of the monoclonal antibodies. A covalent bond is formed, with the aid of EDC, between the carboxyl groups on the particle and the amino group(s) on the antibody. Both methods have been shown to be highly sensitive. While not all of the antibody attached to the particles is done covalently, this method offers superior stability, and because of this the covalent method for attachment of antibodies was used. In order to significantly simplify the assay procedure, a series of optimization steps were explored. These optimizations included development of a direct lysis buffer, reduction in immunocapture time, reduction in the time and temperature required for direct lysis, and performing the reverse transcription and amplification procedures using a single addition of reagents. The original procedure for isolating the genomic RNA was cumbersome and time consuming because it used conventional molecular biology techniques. After immunocapture, the viral membranes were disrupted using relatively high concentrations of SDS. Proteinase K was included to digest any proteins associated with the viral RNA. Purification of the HIV-1 RNA was accomplished using a combination of organic solvent extractions, followed by an overnight ethanol precipitation. A direct lysis buffer was formulated to extract genomic HIV-1 RNA from the intact virion thereby providing for the direct use of the genomic HIV-1 RNA in a reverse transcription and amplification procedure. This eliminated both the organic solvent extraction and the ethanol precipitation procedures that are required in the conventional procedure. At a minimum, a total of 16 hours was eliminated from the assay by not using the organic solvent purification procedure. To determine the amount of reagents needed for direct lysis, the approximate detergent and/or Proteinase K concentrations required to disrupt an equivalent amount of cellular and viral material were determined. Typically, the disruption of 10 6 human cells requires one milliliter of a solution containing 0.25% SDS and 0.5 mg/ml Proteinase K (19). The theoretical binding capacity of the immunoparticles was determined in an earlier study to be approximately 10,000 virions (Henrard, D.R., Mehaffey, W.F., and Allain, J.P. A Sensitive Viral Capture Assay For The Detection Of Plasma Viremia In HIV Infected Individuals. AIDS Res. Human Retro. 1992;8:47-51). Thus, the concentrations of SDS and Proteinase K needed to lyse 10 6 cells should be at least 100 fold greater than that needed to lyse 10,000 virions. Furthermore, the volume of a cell is approximately 1000 times greater than that of a virus (i.e., the average diameters of a cell and a virus are 3 μ and 0.1 μ, respectively). Therefore, a total 10 5 fold reduction in the amount of detergent and Proteinase K should be sufficient to lyse an equivalent amount of viral material. The volume of direct lysis buffer that is added to the immunocaptured virions is, however, 30 μl, which corresponds to a 33 fold decrease in the net amount of detergent and Proteinase K compared to that needed for lysing cells. This would suggest a 3000 fold decrease in the concentration of detergent and/or Proteinase K needed to disrupt the 10,000 virions (10 5 divided by 33), corresponding to SDS and Proteinase K concentrations of -approximately 0.0001% and 0.167 μg/ml, respectively. These concentrations are 10 fold lower than the actual concentrations tested, which gave results comparable to the organic extraction procedure. Thus, the concentrations of SDS and Proteinase K that were used to disrupt the HIV-1 virions were at least 10 fold greater than is required based on these calculations. Neither reverse transcription nor amplification were inhibited when lysis buffers contained Triton X-100, Tween 20, or low concentrations of SDS. No inhibitory effect was seen in lysis buffers containing as high as 0.5% Triton X-100 or 0.45% Tween 20. Interestingly, it appeared that SDS inhibits the reverse transcription reaction at concentrations greater than 0.007%. When the detergent concentration of the direct lysis buffer examined was at 0.01%, its final concentration during the reverse transcription reaction was 0.007%. This concentration decreased further to 0.00147% during the PCR reaction. As previously stated, SDS did not inhibit the PCR at concentrations below 0.001%. It was not determined whether 0.00147% SDS inhibits the reaction, although it does not seem likely that this increase would have a dramatic affect on the PCR reaction. Instead, the lack of a positive result suggests that the reverse transcription reaction was inhibited. This was unexpected because there have not been any reports of low SDS concentrations having an inhibitory effect on the reverse transcriptase enzyme. It is possible that this concentration of SDS somehow complexed with the RT, thereby inhibiting complete or sufficient production of cDNA. Alternatively, SDS may inhibit the reverse transcriptase itself, perhaps by indirectly affecting the structure of its active site. Inhibition of any portion of the RT reaction would have a greater effect on the final amplification of the HIV-1 cDNA, because without the cDNA template, no PCR products would result. The addition of Proteinase K to the direct lysis buffer did not have any adverse effects on either of the enzymatic reactions. In fact, the addition of 1 μg/ml to buffers containing each of the detergents tested gave similar results to the organic extraction procedure. Reduction of the immunocapture time and temperature required for direct lysis had no adverse effect on the sensitivity of the assay. Because the capture time could be reduced from three hours to one hour, it is desirable that the monoclonal antibodies used have an extremely high affinity to the gp120 and gp41 proteins that are associated with the virions. A short incubation time at low temperature was sufficient to disrupt the HIV virions and isolate the HIV-1 RNA, indicating that lysis was very fast and that the detergent and Proteinase K concentrations selected were sufficient to completely dissociate HIV RNA from intact virions. Combining the reverse transcription and amplification procedures into one assay did not have an adverse effect on assay sensitivity. Just as with other targets, (i.e., HCV), performing the RT and PCR reactions with a single addition of reagents was accomplished by optimizing some of the component concentrations in the assay. However, it was surprising to find that the 50 μl single step RT/PCR procedure did not work when samples were assayed. The 50 μl procedure did give comparable assay signals when the RNA and DNA controls were run. All component concentrations, except for the final SDS and glycerol concentrations, remained the same in both procedures. The SDS concentration doubled when the final assay volume was 50 μl (from 0.00015% to 0.0003%). However, this concentration has not been reported to be inhibitory to the Taq polymerase enzyme. Additionally, the glycerol concentration, resulting from the addition of the stock enzymes, in the 50 μl procedure increased over two fold (2.2% vs 0.97%) compared to the 100 μl procedure. According to the manufacturers, no significant inhibitory effect on the enzymes occur when the glycerol concentration is within this range. The decrease in assay volume did, however, concentrate the particles that were associated with the immunocapture. It is possible that the particles somehow either interfered or inhibited the enzyme reactions. Probably the two most important benefits resulting from optimization of the immunocapture procedure were the reduction in the total number of steps involved and the amount of time required to complete the assay. These changes in the assay procedure may significantly reduce the risk of contamination by sample carryover. Table 5 details the differences in procedure between the convention methodologies and the present invention. The use of a direct lysis buffer, the elimination of the organic solvent extraction procedure, and the single step RT/PCR procedure all significantly reduced the number of times each assay tube had to be opened. For example, the actual number of times each tube had to be opened after the immunocapture step went from ten in the standard method to three in the modified method. Additionally, the number of times a reagent was either removed or added to an assay tube went from 15 in the standard method to 4 in the modified method. The major difference in the actual laboratory time required to complete the standard assay procedure and the modified procedure (single step RT/PCR) was in the overnight RNA precipitation step. The elimination of this step from the modified procedures saved, at a minimum, 15 hours. Overall, the modified procedure reduced the immunocapture time by two hours, the time for lysis and isolation of the HIV-1 genomic RNA by 15 hours, and the time to set up the reverse transcription and PCR mixes by another one hour. While at least two working days were required to obtain the results of a sample using the standard procedure, it took only nine hours to get that same result using the modified procedure. Table 5 Comparison Of Standard And Single Step RT/PCR Procedures. Standard Protocol Single Step RT/PCR immunocapture of ΗIV-1 virions immunocapture of HIV-1 virion centrifuge, remove supernate centrifuge, remove supernate wash particles wash particles centrifuge, remove supernate centrifuge, remove supernate add 200 μl SDS/Proteinase K solution, add 30 μl DLB and incubate at 37° C incubate for 1 Hr at 56°C for 30 minutes phenol extract, centrifuge & transfer place two 15 μl samples into clean aqueous phase to new tube ' tubes add equal volume of CHCI3, vortex and denature at 95° C for 5 minutes centrifuge transfer aqueous phase to new tube add 85 μl RT/PCR mix, oil and RT, containing acetate denature, amplify add two volumes of EtOH, vortex and precipitate RNA O/N at -20°C Pellet RNA wash with ice cold 70% EtOH centrifuge, remove supernate redissolve in 30 μl H2O place duplicate 15 μl samples into centrifuge tubes add 5 μl RT mix to each tube heat at 95°C for 5 minutes add 1 μl of RT/RNasin RT at 37° C for 30 minutes add 30 μl of water and 50 μl of oil, denature at 95°C for 5 minutes add 50 μl of PCR mix and amplify The strict adherence to contamination control procedures aided in controlling the occurrence of false positive reactions. This was accomplished by using three separate laboratories to do sample preparation, PCR and detection. Each laboratory contained separate sets of equipment. The routine use of barrier pipet tips and UV sterilization was also implemented to reduce and possibly eliminate contamination due to amplification products. Although significant measures were undertaken to control contamination, false positive reactions did occur at a rate of approximately 1 per 500 tests. This indicates that adherence to contamination control procedures are critical in order to maintain the high specificity of PCR assays. The simplified immunocapture-cDNA/PCR was then used to determine whether there was an association between HIV-1 plasma viral load and the likelihood of either vertical or horizontal HIV-1 transmission. The women included in the HIV-1 vertical transmission study were relatively healthy and showed no symptoms of disease. Their mean CD4+ counts were high (178 to 1113 mm 3 ) and beta2 microglobulin levels were within normal values (average 1.64 μg/ml), suggesting that the women included in the study were relatively healthy. Positivity for HIV-1 RNA (plasma viremia) correlated with lower CD4+ counts. This was consistent with earlier studies, which had shown that there was a correlation between CD4+ count and increased plasma viremia (Saag, M.S., Crain, M.J., Decker, W.D., Campbell-Hill, S., Robinson, S., Brown, W.E., Leuther, M., Whitley, R.J., Hahn, B.H., and Shaw, G.M. High-Level Viremia In Adults And Children Infected With Human Immunodeficiency Virus: Relation To Disease Stage And CD4+ Lymphocyte Levels. J. Infect. Pis. 1991;164:72-80). In contrast, no correlation was found between the level of plasma HIV-1 viral load and vertical transmission. Other reports have documented a lack of maternal plasma viremia with vertical transmission (Ariyoshi, K., Weber, J., and Walters, S. Contribution Of Maternal Viral Load TO HIV-1 Transmission. Lancet 1992;340; Puel, J., Izopet, J., Lheritier, D., Briant, L., Guyader, M., Tricoire, J. and Berrebi, A. Viral Load And Mother To Infant HIV Transmission. Lancet 1992;340:859-860). It has been suggested that the occurrence of maternal virulent fast-replicating HIV variants (Grunters, R.A., Terpstra, F.G., De Goede, R., Mulder, J.W., De Wolf, F., Schellekens, P., Van Lier, R., Termette, M., and Miedema, F. Immunological And Virologic Markers In individuals Progressing From Seroconversion To AIDS. AIDS 1991;5:837-844), the selective transmission of maternal variants (Wolinsky, S.M., Wike, C.A., Korber, B., Hutto, C., Parks, W.P., Rosenblum L.L., Kunstman, K.J., Furtado, M.R., and Munoz, J.L. Selective Transmission Of Human Immunodeficiency Virus Type-1 Variants From Mothers To Infants. Science 1992;255:1 134-1 137), and co-infection with other microbial agents (Holmes, W. Vertical Transmission Of HIV. Lancet 1991 ;337:793-794) may be important variables in the transmission of HIV-1 from a mother to her child. Our results also suggest that factors other than viral load may contribute to the vertical transmission of HIV-1. Trauma to the placenta may result in both cellular and viral entry into the developing fetus. The possibility of infection while the child is passing through the birth canal also seems plausible, depending on the amount of maternal blood present during delivery. Entry of the virus into the child could occur through ingestion, the lacrimal glands, or through small cuts that may occur during delivery. In those cases, the amount of maternal blood lost during delivery may be more important than the actual viral load. A woman with a low viral load but extensive blood loss maybe more likely to transmit HIV than a woman with high viral load but minimal blood loss during delivery. This may explain why, in our study, an asymptomatic mother who had no detectable HIV RNA in her peripheral blood, and a very high CD4+ count, could still transmit HIV-1 to her child. Unlike vertical HIV-1 transmission, the horizontal transmission of HIV-1 from a blood donor to a recipient was highly correlated with plasma viremia. All twenty-two blood recipients in this study were transfused with HIV-1 seropositive blood. A significant proportion (64%) of the donor samples that transmitted an HIV-1 infection to the recipient had detectable HIV-1 viremia, while the individuals that did not infect the recipient had no detectable HIV-1 viremia. These results suggest that HIV infection depends primarily on the level of plasma viremia in the context of blood transfusions. As the number of HIV-1 infected individuals increases, it is becoming more important to monitor their status during the course of infection. The development of an assay that can rapidly and efficiently identify the progression of the disease will aid in monitoring the infection, and the efficacy of various therapeutics. The immunocapture -cDNA/PCR assay, which is highly sensitive, may be particularly useful for these purposes. The assay described here can differentiate log differences in plasma HIV-1 viral load and efforts are currently underway to develop a more precise way of detecting and quantitating the level of plasma HIV-1 viremia. These include the development of a quantitative detection system based on an amplification system other than the polymerase chain reaction. Besides PCR, there have been two other methods described in the literature that are currently being used to amplify target DNA. One is called Self Sustained Sequence Replication (3SR) and the other is the Ligase Chain Reaction (LCR) (Bush, C.E., Donovan, R.M., Peterson, W.R., Jennings, M.B., Bolton, V., Sherman, D.G., Vanden Brink, K.M., Beninsig, L.A., and Godsey, J.H. Detection Of Human Immunodeficiency Virus Type 1 RNA In Plasma Samples From High Risk Pediatric Patients By Using The Self Sustained Sequence Replication Reaction. J. Clin. Micro. 1992;30:281-286; Wu, D.Y. and Wallace, R.B. The Ligation Amplification Reaction (LAR)- Amplification Of Specific DNA Sequences Using Sequential Rounds Of Template- Dependent Ligation. Genomics 1989;4:560-569; Barringer, K.J., Orgel, L., Wahl, G., and Gingeras, T.R. Blunt-end And Single Strand Ligations By Escherichia coli Ligase: Influence On An In Vitro Amplification Scheme. Gene 1990;89:117-122). The ligase chain reaction uses a thermostable DNA ligase to covalently join adjacent 3' hydroxyl and 5' phosphoryl termini of the oligo primers that are complementary to the target DNA, and Taq polymerase is not required. Like PCR, the ligase chain reaction amplifies the target DNA by use of a series of annealing and denaturation steps. The oligonucleotide products from each round serve as substrates for each successive round. This makes it possible to increase the number of target molecules of DNA by a factor of over 10 5 fold. To detect the products of LCR, the primers are modified to include a fluorophore. An automated fluorimetric assay system is then used where 24 samples can be processed in 45 minutes. BIBLIOGRAPHY ( 1 ) Centers for Disease Control. HIV Prevalence Estimates And AIDS Case Projections For The United States: Report Based Upon A Workshop. MMWR 39:(no.RR-16) November 30, 1990. ( 2 ) The European Collaborative Study. 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Claims: CLAIMS 1 . A lysis reagent comprising a detergent and Proteinase K in concentrations compatible with the enzyme reactions used in reverse transcription and nucleic acid amplification procedures and wherein said detergent is selected from the group consisting of sodium dodecyl sulfate, Triton X-100 and Tween 20. 2. The reagent according to Claim 1 , wherein said detergent is sodium dodecyl sulfate at a concentration less than or equal to 0.001%. 3. The reagent according to Claim 1, wherein said detergent is selected from the group consisting of Triton X-100 and Tween 20 at a concentration ranging from 0.1- 0.5%. 4. A composition comprising a buffer, 0.001% sodium dodecyl sulfate and one microgram/milliliter Proteinase K for use as a direct lysis buffer in the isolation of nucleic acids. 5. The composition according to Claim 4, wherein said buffer is 10 mM TRIS (pH 7.0) .

Inventors: HENRARD DENIS R, PHILLIPS JACK, HENRARD, DENIS, R., PHILLIPS, JACK
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1994
Main classification: C11D17
IPC codes: C11D1, C11D3, C12N1, C12N9, C12N15, C12Q1